Human Anaplastic Large Cell Lymphomas (ALCL) are a unique subset of lymphomas partly distinguished by their co-expression of the CD30 antigen (Stein, H. et al. (2000), Blood 96:3681-3695). Classical cytogenetic studies demonstrated that ALCLs carry unique translocations within the p23 region of chromosome 2, (Rimokh R., et al. carry unique translocations within the p23 region of chromosome 2, (Rimokh R., et al. (1989), Br J Haematol. 71:31-36. Kaneko Y., et al. (1989), Blood. 73:806-813 and Le Beau, M M et al. (1989) Leukemia. 3:866-870). In 1994, Morris et al. cloned the t(2;5) translocation and discovered that a novel tyrosine kinase gene, the Anaplastic Lymphoma Kinase (ALK), was fused to the NPM/B23 gene (Morris, S W et al. (1994) Science. 263:1281-1284). NPM participates in nucleocytoplasmic trafficking (Wang D., et al. (1993) Cell Mol Biol Res. 39:33-42 and Szebeni A. et al. (1999) Protein Sci. 8:905-912) and has been recently shown to regulate the duplication of centrosomes (Okuda, M. et al. (2000) Cell. 103:127-140). The ALK gene encodes a tyrosine kinase receptor whose physiological expression is largely limited to neuronal cells (Iwahara, T. et al. (1997) Oncogene. 14:439-449 and Morris, S W, et al. (1997) Oncogene 14:2175-2188). However, the physiological role of the ALK receptor remains largely unknown since ALK−/− mice appear normal (Duyster, J. et al. (2001) Oncogene. 20:5623-5637). Nonetheless, ALK is phylogenetically highly conserved (Iwahara, T. et al. (1997) Oncogene. 14:439-449 and Morris, S W, et al. (1997) Oncogene. 14:2175-2188), suggesting that it might have an important role in neuronal cellular function. In fact when constitutively activated in the rat pheocromocytoma cells PC12, ALK leads to neuronal differentiation and provides anti-apoptotic signals in stress conditions (Souttou, B. et al. (2001) J Biol Chem. 276:9526-9531). (Piva et al. personal communication). Recently, Stoica et al. have also demonstrated that pheotrophin binds to ALK receptor (Stoica, G E, et al. (2001) J Biol Chem. 276:16772-16779), but other additional ligands might exist.
In the past five years, several groups have successfully cloned new ALCL translocations and demonstrated that the ALK gene can fuse to multiple targets, which include the TFG, TPM3, ATIC, CLTCL, RanBP2 and MSN genes (Duyster, J. et al. (2001) Oncogene. 20:5623-5637). Proteins fused to ALK largely determine the subcellular localization of the derived fusion proteins, being cytoplasmic (ATIC-, TGF-ALK etc.), cytoplasmic and nuclear (NPM-ALK), or membranous (MSN-ALK) (Duyster, J. et al. (2001) Oncogene. 20:5623-5637). Moreover, ALK translocations can also be detected in non-lymphoid neoplasms such as inflammatory myofibroblastic tumors (Coffin, C M. et al. (2001) Mod Pathol. 14:569-576), and ALK expression has been described in neuroblastomas (Lamant, L. et al. (2000) Am J Pathol. 156:1711-1721), as well as in a unique subtype of IgA positive plasmacytoid tumors (Delsol, G. et al. (1997) Blood. 89:1483-1490).
Cellular transformation by NPM-ALK has been demonstrated in immortalized rodent fibroblasts (Bai, R Y. et al. (1998) Mol Cell Biol. 18:6951-6961), and confirmed in studies which have shown that ALK protects Ba/F3 and PC12 cells from interleukin-3 or growth factor withdrawal (Stoica, G E., et al. (2001) J Biol Chem. 276:16772-16779 and (Bai R Y., et al. (1998) Mol Cell Biol. 18:6951-6961). (Piva et al. personal communication). Transfer of NPM-ALK transduced bone-marrow cells into irradiated host recipient mice resulted in the generation in vivo of large cell B cell lymphomas (Kuefer, M U. et al. (1997) Blood. 90:2901-2910). In the past few years, the molecular mechanisms of NPM-ALK-mediated cellular transformation have also been partially elucidated (Duyster, J. et al. (2001) Oncogene. 20:5623-5637). It has been shown that the ALK portion of the fusion protein, corresponding to the cytoplasmic tail of the ALK receptor and containing the catalytic domain, is absolutely required for transformation (Bai, R Y. et al. (1998) Mol Cell Biol. 18:6951-6961), whereas all the N-terminal regions of the ALK chimeras function as dimerization domains (Bischof, D. et al. (1997) Mol Cell Biol. 17:2312-2325) and (Duyster, J. et al. (2001) Oncogene. 20:5623-5637). As a result of spontaneous dimerization, ALK undergoes autophosphorylation and becomes catalytically active. Constitutively active ALK fusion proteins can bind multiple adaptor proteins and activate a series of pathways involved in cell proliferation, transformation and survival. These include the PLC- Shc PI3-K/Akt and the Jak3-Stat3 pathways (Bai, R Y. et al. (1998) Mol Cell Biol. 18:6951-6961; Bai R Y., et al. (2000) Blood. 96:4319-4327 and Zamo, A. et al. (2002) Oncogene. 21:1038-1047). All these molecules and their putative roles were identified using either non-hematopoietic cells or immortalized B cells, leaving the molecular mechanisms of T cell transformation by ALK chimeras still unknown.
Transgenic animals are among the most useful research tools in the biological sciences. These animals have a heterologous (i.e., foreign) gene, or gene fragment, incorporated into their genome that is passed on to their offspring. Although there are several methods of producing transgenic animals, the most widely used is microinjection of DNA into single cell embryos. These embryos are then transferred into pseudopregnant recipient foster mothers. The offspring are then screened for the presence of the new gene, or gene fragment. Potential applications for transgenic animals include discovering the genetic basis of human and animal diseases, generating disease resistance in humans and animals, gene therapy, drug testing, and production of improved agricultural livestock.